DEPARTMENT OF CONSERVATIVE

DENTISTRY AND
ENDODONTICS

SEMINAR

SEM IN ENDODONTICS
PRESENTED BY
DR. RASHMI SOLANKI
M.D.S IST YEAR
GUIDED BY
DR. MANDEEP.S.GREWAL
DR. AMIT GANDHI
DR. VANDANA BHARDWAJ

PDM DENTAL COLLEGE


Contents
1. Introduction
2. History
3. Construction of SEM
4. Basics of specimen preparation
5.Why images are visible
6. SEM use in endodontics

History

- The development of a SEM began a few yrs after the invention of a TEM by
Ruska - In 1931, but the commercialization of the SEM required about 30
yrs.
In 1935, the original prototype of the SEM, which scans the specimen with an
e- beam to obtain an image, was made by Knoll(Germany).
In 1942, Zworykin (USA), developed a SEM for observing a bulk specimens.
In 1965, Cambridge Scientific Instrument (UK) & JOEL (Japan) first
commercialized SEM individually.

Construction of SEM
A) Electron optical system.(to produce e-) electron gun, condenser lens, objective
lens, scanning coil.
B) Specimen stage (to place the specimen).
C) Secondary e- detector (to collect secondary e-).
D) Image display unit.
E) Operating system. The electron optical system and a space surrounding the
specimen are kept at vacuum.
Electron Gun- it produces an e- beam.
. It is a thermionic emission gun. (TEG), the thermo electrons are emitted from a
filament (cathode) made of a thin tungsten wire, (about 0.1mm ) by heating the
filament at high temperature (about 2800k). Tungsten filament assembly
These e- are gathered as an e-beam, flowing into the metal plate (anode) by
applying a positive voltage.
If the hole is made at the center of the plate the e- beam flows through this hole. If
an electrode (Wehnelt electrode)is placed between the cathode and anode and applied
with the negative charge, the speed of the e- beam can be adjusted.

 Condenser lens/Electromagnetic lens- placing a lens below the e- gun enables to

adjust the diameter of the e- beam.
A fine e- beam is requires for SEM . A typical Electro Magnetic Lens.

Role of the condenser lens in formation of fine e- beam

The aperture is placed between the condenser lens and objective lens.
The aperture made of a thin metal plate, has a small hole.
The e- beam, which passed through the condenser lens, illuminates this apertureplate.
The aperture allows a part of the e- beam to reach the objective lens.
If the excitation of the condenser lens is increased, the e- beam greatly broadens on
the aperture and therefore the number of the electrons reaching the objective lens
decreases.
To the contrary, if the excitation of the condenser lens is decreased, the e- bean
doesnt broaden very much and therefore, most of the electrons pass through the
aperture and many electrons reaches the objective lens.
In this way the e- probe diameter and the probe current can be adjusted.
Objective lens- the objective lens is used for focusing.
Specimen stage- It supports the specimen.
Moves smoothly.
It can perform horizontal (x,y axis) & vertical movements(z axis), tilting of
specimen, and rotation.
The horizontal movement is used for selection of the field while the vertical
movement is used to change the image resolution. SEM opened sample chamber

Secondary e- detector- It is used for detecting the secondary e- emitted from the
specimen.
A scintillator (fluorescent substance) is coated on the tip of the detector and a high
voltage of about 10 kV is applied to it.
The secondary e- are attracted to this high voltage and then generate light when
they hit the scintillator.
This light is directed to a photon-multiplier tube (PMT) through a light guide.
The light is converted to the electrons, and these are amplified as an electric signal.
Secondary electron detector Everhard -Thornley Detector
A supplementary electrode, called the collector, is placed before the scintillator,
and is applied with few hundreds of voltage.
This collector, helps the scintillator to acquire secondary electrons.

 By changing the voltage the amount of secondary electrons to be collected can be

adjusted.
This collector was originally developed by Everhart and Thornley, so this detector
can also be called as E-T detector.
When SEM is equipped with a strongly excited objective lens for higher resolution
a secondary electron detector is placed above the objective lens.
This type of detector is called TTL ie. (Through The Lens ) detector.
Image display unit- The out put signals from the secondary electron detector are
amplified and then transferred to the display unit.
Since the scanning on the display unit is synchronized with the electron-probe
scan, brightness variation, which depends on the number of the secondary electrons,
appears on the monitor screen on the display unit, thus forming a SEM image.
In general, the scan speed of the electron probe can be changed in several
steps.
An extremely fast speed is used for observation and a slow scan speed is used for
saving of image.
The image is recorded in a digital format (electrical file), It is easier to process
image and convenient to send or receive image information.
Vacuum system
- The electron optical system and the specimen chamber must be kept at a high
vacuum of 10-3 to 10-4 Pa.
Thus , these component is evacuated by diffusion pump. If a user desire an oil-free
environment, a turbo molecular pump may be used.
Sample Preparation
1. Cleaning the surface of the specimen.
2. Stabilizing the specimen.
3. Rinsing the specimen.
4. Dehydrating the specimen.
5. Drying the specimen.
6. Mounting the specimen.
7. Coating the specimen.

Cleaning the surface of the specimen.

The proper cleaning of the surface of the specimen is important, because the surface
contains many unwanted deposits, such as dust, mud, soil etc. depending upon the
source of the sample/specimen.

 Stabilizing the specimen.

Hard, dry materials such as wood, bone, feathers, dried insects, or shells can be
examined with little further treatment, but living cells and tissues and whole, softbodied organisms usually require chemical fixation to preserve and stabilize their
structure.
Stabilization is typically done with fixatives. Fixation cab be achieved by :1. Perfusion or microinjection.
2. Immersions.
3. With vapours.
Fixation is usually performed by incubation in a solution of a buffered chemical
fixative, such as glutaraldehyde, sometimes in combination with formaldehyde and
other fixatives. Fixatives that can be used are:1. Aldehydes.
2. Osmium tetroxide.
3. Tanic acid.
4. Thiocarbohydrazides.
Rinsing the specimen.
After the fixation step, the sample must be rinsed in order to remove excessive
fixatives.
Dehydrating the specimen.
All water must be removed from the samples because the water would vaporize in
the vacuum.
The fixed tissue is then dehydrated. Because air- drying causes collapse and
shrinkage, this is commonly achieved by replacement of water in the cells with
organic solvents such as ethanol or acetone.
Dehydration is performed with a graded series of ethanol or acetone.
Drying the specimen.
For SEM, a specimen is normally required to be completely dry, since the
specimen chamber is at high vacuum.
Otherwise the sample will be destroyed in the electron microscope chamber.
Mounting the specimen.

 After the specimen has been cleaned, fixed, rinsed, dehydrated and dried, using an
appropriate protocol, specimen has to be mounted on the holder that can be inserted
into the scanning electron microscope.
All samples must also be of an appropriate size to fit in the specimen chamber and
are generally mounted rigidly on a specimen holder called a specimen stub.
The dry specimen is usually mounted on a specimen stub using an adhesive such
as epoxy resin or electrically conductive double-sided adhesive tape.
Charge-up
This charge-up phenomenon can be prevented by coating the non-conductor
sample with metal (conductor).
Sample coating is intended to prevent charge-up phenomenon by allowing the
charge on the specimen surface go to ground through the coated conductive film.
Coating the specimen.
The idea of coating the specimen is to increase the conductivity of the specimen
and to prevent the high voltage charge on the specimen by conducting charge to the
ground.
These specimen are coated with thin layer ie.20nm- 30nm of conductive metal.
All metals are conductive and require no preparation before being used.
All non-metals need to be made conductive by covering the sample with a thin
layer of conductive material.
This is done by using a device called a "sputter coater.
Conductive materials in current used for specimen coating includes gold, goldpalladium alloy , platinum, osmium , iridium, tungsten, chromium and graphite.
Sputter Coater

Advantages
1. Thermal conductivity is increased.
2. Damaged to the sample is reduced.
3. The quantity of secondary electrons is increased.
Disadvantages
1. The shape & size of nano particle is lost or altered.
2. Specimen information about elemental composition & surface potential may be
lost. Metal Coating

 Why Images Are Visible?

The SEM image appears as if it is been observed by the naked eye.
Interaction of electrons with specimens: when e- enters the specimen, they are
scattered with in the specimen and gradually lose their energy.
The scattering range of the electrons inside the specimen is different depending on
the electron energy, atomic number, and the density of the constituent atom.
If the atomic number and density are larger, the scattering range is smaller.
If the electron energy is higher then the scattering range is larger.

How Does SEM Works

To further understand how does SEM works, we must begin with the electrons. In
a light microscope, light from a source (usually an incandescent light) is focused
through lenses onto the sample. The image is formed when the sample reflects and
absorbs different wavelengths of this light which is detected by our eyes and formed
into an image by our brains. An electron microscope works in a similar fashion.
Electrons from a source are focused on the sample. These electrons reflect off the
sample, they are then picked up by an electron detector and then processed into an
image which is projected onto a CRT that our eyes can see.
To begin our understanding of how an SEM works, let's begin with the source of
electrons, the electron gun.
Most SEMs have what is called a hot cathode source, usually a tungsten filament
similar to that in a light bulb.
When such a filament is heated by passing current through it, it not only emits
light, but an electron cloud forms around the filament.
Left on their own, they remain in the cloud and are reabsorbed into the filament
when the current is removed.
Placing a positively charged plate (an anode) near the filament and the electrons
(being negatively charged) will be attracted to it.
Problem is, the electrons would not be well directed and would probably jump
over to the anode plate in a series of arcs.
But by placing a negatively charged cathode plate near the filament (which they
are repelled by) with a hole in it and a positively charged anode (which they are
attracted to) under this with another hole in it and we have the makings of an electron
gun.
The electron cloud is attracted to the anode plate enough that they will travel
through the hole in the cathode. But in doing so, they gain enough speed that most of
them travel right through the hole in the anode plate.
Now we have an electron gun. The speed of the electrons emitted from this gun is
controlled by the amount of potential (voltage) applied to the cathode and anode
plates.

 The electrons from the gun come out in almost a spray pattern, so we may have a
flow of electrons, but this could hardly be called a beam.
We need lenses to control the flow of electrons, however, the glass lenses of a light
microscope will not work. Instead, an electron microscope uses electromagnetic
lenses. spray pattern

An electromagnetic lens An electromagnetic lens is a relatively simple device.

By applying current to wire coiled around an iron cylindrical core, a magnetic field is
created which acts as a lens.
The advantage of an electromagnetic lens in an electron microscope is that by varying
the current through the wires, the lens can have a variable focal length.

We now can arrange the electron gun and lenses in a column mounted on a
sample chamber.
The condenser lens controls the size of the beam, or the amount of electrons
traveling down the column.
The objective lens focuses the beam into a spot on the sample. This is necessary to
have an image in proper focus.

So far, the column we have designed will just focus the electron beam into a
spot on the sample.
This is fine for welding or if we wanted the beam to pass through the sample as in
a TEM, but for an SEM to work, we need the beam to scan.
By placing sets of plates around the beam and varying the potential between them,
the electron beam can be deflected. If these plates are attached to a scan generator, the
beam can be made to scan lines across the sample
But this scan generator is not only controlling the scan coils, but is also
controlling the beam of a CRT, the image formed on the CRT will be synched to the
electron beam scanning the sample.
So now we have a beam that is scanning across the sample surface and this beam
is synched to the beam of a CRT.

But how is the image formed?

 To understand this, we need to know what happens when the electron beam
interacts with the atoms of the sample.
The incident beam electrons (from the electron gun) do not simply reflect off the
sample surface.
As the beam travels through the sample it can do three things:
1. It can pass through the sample without colliding with any of the sample atoms
(matter is mostly space).
2. It can collide with electrons from the sample atoms, creating secondary electrons.
3. It can collide with the nucleus of the sample atom, creating a backscattered
electron.

How secondary e- are formed

The incident beam is composed of highly energized electrons. If one of these
electrons collides with a sample atom electron, it will knock it out of its shell. This
electron is called a secondary electron and is weak in energy. If these secondary
electrons are close enough to the sample surface, they can be collected to form a SEM
image.
The incident beam electron loses little energy in this collusion. In fact, a single
electron from the beam will produce a shower of thousands of secondary electrons
until it doesn't have the energy to knock these electrons from their shells.

How backscattered e- are formed

If the incident beam collides with a nucleus of a sample atom, it bounces back out
of the sample as a backscattered electron.
These electrons have high energies and because a sample with a higher density will
create more of them, they are used to form backscattered electron images, which
generally can discern the difference in sample densities.
Are used to determine crystal structures and orientations of minerals
An electron detector is placed in the sample chamber. By having a 10 keV positive
potential on its face, it attracts the secondary electrons emitted from the sample
surface.

Detection of Secondary Electrons

 Secondary electrons hit against the scintillator for conversion into the optical
signal, which are reconverted into electrons on the photoelectric conversion face .
These electrons are accelerated with the electric field and hit against the first
dynode.
These electrons are then led to next dynode to produce a large number of
secondary electrons.
Thus the number of secondary electrons increases sequentially and finally then
taken out as a signal current.

So how is the contrast formed?

In secondary imaging mode, as the incident beam scans across the sample's surface
topography, secondary electrons are emitted from the sample.
If the beam travels into a depression or hole in the sample, the amount of
secondary electrons that can escape the sample surface is reduced and the image
processing places a corresponding dark spot on the screen.
Conversely, if the incident beam scans across a projection or hill on the sample,
more secondary electrons can escape the sample surface, and the image processing
places a bright spot on the screen.
This form of image processing is only in gray scale which is why SEM images are
always in black and white.
These images can be colorized through the use of feature-detection software, or
simply by hand editing using a hand graphic editor.
This is usually for aesthetic effects, for clarifying structure, or for adding a realistic
effect to the sample.

In backscattered imaging mode, as the incident beam scans across the sample's
surface topography, backscattered electrons are emitted from the sample.
A low atomic weight area of the sample will not emit as many backscattered
electrons as a high atomic weight area of the sample.
In reality, the image is mapping out the density of the sample surface.

So how does a SEM change the magnification of an image?

By reducing the size of the area scanned by the scan coils, the SEM changes the
magnification of the image.
Secondary image showing surface morphology Backscattered image showing
compositional inhomogeneity Image of a cement
Light region is made up predominantly of Fe. (i.e. the heaviest element) Grey
region is made up predominantly of Ca. Dark region is made up predominantly of Si
and Al. (i.e. the lightest elements)

 SEM Applications
SEMs have a variety of applications in a number of scientific and industry-related
fields, especially where characterizations of solid materials is beneficial.
In addition to topographical, morphological and compositional information, a
Scanning Electron Microscope can detect and analyze surface fractures, provide
information in microstructures, examine surface contaminations, reveal spatial
variations in chemical compositions, provide qualitative chemical analyses and
identify crystalline structures.

Presence of extensive areas of resorption can be noticed on the lingual aspect of all
roots.

(A-D)- Root resorption patterns of a permanent molar (13 and 100x) and a permanent
incisor (20 and 150x)
Ravindran Sreeja,Chaudhary Minal; Tumsare Madhuri; Patil Swati; Wadhwan Vijay J.
Appl. Oral Sci. vol.17 no.5 Bauru Sept./Oct. 2009 A scanning electron microscopic
study of the patterns of external root resorption under different conditions

Mandibular permanent molar undergoing root resorption due to an associated

periapical granuloma

(A-D)- Root resorption of a premolar subjected to orthodontic force at 14x, 100x,

250x, 500 magnifications
Ravindran Sreeja,Chaudhary Minal; Tumsare Madhuri; Patil Swati; Wadhwan Vijay J.
Appl. Oral Sci. vol.17 no.5 Bauru Sept./Oct. 2009 A scanning electron microscopic
study of the patterns of external root resorption under different conditions

Mandibular first premolar undergoing pressure resorption during the course of

orthodontic treatment

(A-D)- Root resorption of a premolar subjected to orthodontic force at 14x, 100x,

250x, 500 magnifications
Ravindran Sreeja,Chaudhary Minal; Tumsare Madhuri; Patil Swati; Wadhwan Vijay J.
Appl. Oral Sci. vol.17 no.5 Bauru Sept./Oct. 2009 A scanning electron microscopic
study of the patterns of external root resorption under different conditions

Human blood was obtained by venous puncture.

 The RBCs were isolated by centrifugation.

Fixation- 1% glutaraldehyde
Washed- phosphate buffer.
Mounting
Sputter coating- with gold

SEMs can be as essential research tool in fields such as life science, biology,
gemology, medical and forensic science, metallurgy.
In addition, SEMs have practical industrial and technological applications such as
semiconductor inspection, production line of miniscule products and assembly of
microchips for computers.

SEM Advantages
Advantages of a Scanning Electron Microscope include its wide-array of
applications, the detailed three-dimensional and topographical imaging and the
versatile information garnered from different detectors.
SEMs are also easy to operate with the proper training and advances in computer
technology and associated software make operation user-friendly. This instrument
works fast.
SEM Disadvantages
The disadvantages of a Scanning Electron Microscope start with the size and cost.
SEMs are expensive, large and must be housed in an area free of any possible
electric, magnetic or vibration interference.
Maintenance involves keeping a steady voltage, currents to electromagnetic coils
and circulation of cool water.

 Special training is required to operate an SEM as well as prepare samples.

The preparation of samples can result in artifacts. The negative impact can be
minimized with knowledgeable experience researchers being able to identify artifacts
from actual data as well as preparation skill. There is no absolute way to eliminate or
identify all potential artifacts.
In addition, SEMs are limited to solid, inorganic samples small enough to fit inside
the vacuum chamber that can handle moderate vacuum pressure.
Finally, SEMs carry a small risk of radiation exposure associated with the electrons
that scatter from beneath the sample surface.

In Endodontics, SEM is used mainly to evaluate bacterial leakage within the root
canal, bacterial biofilm formation and also to evaluate fracture patterns regarding root
posts and filling cements. Topographic analysis of the dentin surface after different
rotary instruments and techniques is also a common purpose of study.
SEM is particularly important in Endodontics when the gap formed between the
filling material and the dentin wall is analyzed or measured. According to Souza et al,
replicas should be made and evaluated before samples are prepared for SEM
examination in order to differentiate genuine gaps from artifactual gaps created after
vacuum desiccation in conventional scanning electron microscopes. The usage of
SEM technology in Endodontics allows visualization of root/dentin structures, with
different heights, without altering the focus. In addition, since SEM figures are in gray
scale, the color of dentin does not influence in obtaining a correct focus, limitation
which is found in optical stereomicroscopes.

Dentinal Tubules in SEM

The purpose of this in vitro study was to determine the degree of removal of
pulpal remnants and smear layer from root canals after final irrigation with
three different solutions. During instrumentation the step-back preparation and
1% NaOCl were used. The final 4-min, 30-ml irrigation varied as follows:
group I, 10 ml of 1% NaOCl + 10 ml of 10% citric acid + 10 ml of distilled
water; group II, 15 ml of 0.5% NaOCl + 15 ml of EDTA-T; and group III, 10
ml of 5% NaOCl + 10 ml of 3% H2O2 + 10 ml of 5% NaOCl.
Scanning electron microscopic photomicrographs were evaluated for the mean
number of visible open dentinal tubules by three observers. The largest
number of visible tubules in the three groups was in the cervical third,
followed by the middle and apical thirds.
Miriam F et al Efficacy of Final IrrigationA Scanning Electron Microscopic
Evaluation

 The microscopic details of the de-bonded interfaces between endodontic

sealers and dentin or gutta-percha were assessed in this study. Dentin,
conditioned with 37% H3PO4 for 30 s, 25% citric acid for 30 s, 17% EDTA for
5 min, or a rinse with 10 ml of distilled H 2O (control), and gutta-percha
surfaces were coated with freshly mixed sealer: Grossmans sealer, Apexit,
Ketac-Endo, AH Plus, RoekoSeal Automix, or RoekoSeal Automix with an
experimental primer. The surfaces were pressed together and the sealers were
allowed to set. After tensile bond strength testing, the morphological aspects
of the fractured surfaces were assessed.
Some of the sealers penetrated into the dentinal tubules when the dentin
surface had been pretreated with acids. However, these sealer tags remained
occluding the tubules after bond failure in some instances only (Grossmans
sealer, RoekoSeal Automix with an experimental primer, AH Plus/EDTA).
Penetration of the endodontic sealers into the dentinal tubules when the smear
layer was removed was not associated with higher bond strength.
Iman M. Saleh et al. Adhesion of Endodontic Sealers: Scanning Electron Microscopy
and Energy Dispersive Spectroscopy

The purpose of this scanning electron microscope (SEM) study was to

compare the cleanliness of the root canal walls following either a manual or a
rotary technique of canal instrumentation. Significantly less debris was found
in the apical region using the manual filing technique (P < 0.05); no
significant differences could be found at the other levels. Overall, significantly
less debris was found on the root canal walls using the manual technique when
the data from the three levels were compared (P = < 0.05). The manual
technique employed in this study produced cleaner root canal walls than the
rotary ProFile technique.
M. Ahlquist et al. The effectiveness of manual and rotary techniques in the cleaning of
root canals: a scanning electron microscopy study.

The purpose of this study was to examine the presence of bacteria on the
apical root surfaces of untreated teeth associated with chronic periradicular
lesions. Twenty-seven extracted teeth with extensive carious lesions,
radiolucent lesions of varying sizes and attached periradicular lesions after
extraction, were selected for study. Following fixation, lesions were removed
and the apical 5-mm portion of each root was sectioned. Root tips were
dehydrated, sputter-coated with gold, and then examined for the occurrence of
bacteria on the apical root surfaces using a scanning electron microscope.
Bacterial cells were usually observed close to the apical foramen, but
restricted to the root canal. Morphologically, these bacteria consisted of cocci

and rods. A dense bacterial aggregate composed mainly of rods was observed
within the root canal and surrounding the apical foramen of one specimen.
Beyond the apical foramen, other bacterial morphological types were
recognized, including coaggregations of cocci and filaments, characterizing a
fully developed corn cob. Extraradicular bacteria were observed in one tooth
out of 27 (4% of the cases).
J. F. Siqueira Jr et al. Bacteria on the apical root surfaces of untreated teeth
with periradicular lesions: a scanning electron microscopy study

Debris and smear layer scores after two types of instruments manufactured
from different alloys were used to ultrasonically activate irrigants during canal
preparation. The influence of two rotary preparation techniques on cleanliness
of the shaped canals was also studied. Apical stops were prepared to size 45 in
42 single-canalled extracted premolars and canines, which were divided into
six equal groups. Groups 1, 2 and 3 were prepared by ProFile .04 (PF) while
groups 4, 5 and 6 were prepared by Lightspeed (LS). All groups were irrigated
using 5.25% NaOCl and 17% EDTA. Irrigants in groups 2 and 5 were
ultrasonically activated using a size 15 steel K-file and by a blunt flexible
nickeltitanium wire in groups 3 and 6. Groups 1 and 4 served as negative
controls.
Roots were split and canal walls examined at 15, 200 and 400
magnification in an SEM. Smear layer and debris scores were recorded at 3, 6
and 9 mm levels using a 5-step scoring scale and a 200-m grid. Although all
groups had significantly higher smear layer and debris scores at the 3 mm
levels compared to the 9 mm levels (P < 0.05), no significant differences were
recorded due to the ultrasonic energy transmitted by the two alloys.
Ultrasonically activated irrigants did not reduce debris or smear layer scores.
This finding was not influenced by the material nor by the design of the
instrument used to transmit ultrasonic activation.
B. E. Mayer et al. Effects of rotary instruments and ultrasonic irrigation on debris and
smear layer scores: a scanning electron microscopic study

Summary
In conclusion, although SEM is an extremely important tool for research in dentistry,
researchers should provide full information when using SEM figures, since the
comparison of results is only possible when similar magnifications is used. In
addition, how the sample was processed, regarding conductivity, what type of
microscope was used (tungsten, LaB6 beam microscopes or FEG-SEM) is crucial
information that should also be in the article. Lack of information makes the
understanding of results, as well as the comparison, difficult for any researcher when
using SEM technology.

References
1. Theory & practice of histological techniques: John Bancroft, M Gamble.
2. Scanning Electron Microscopy, Dr H. Bagshaw
3. Introduction to SEM. By Rodney Herring
4. Hortol, P. (2010). "Using digital colour to increase the realistic appearance of
SEM micrographs of bloodstains". Micron 41 (7): 904908.
5. "Introduction to Electron Microscopy" . FEI Company. p. 15. Retrieved 12
December 2012
6. Ravindran Sreeja,Chaudhary Minal. -J. Appl. Oral Sci. vol.17 no.5 Bauru
Sept./Oct. 2009-A scanning electron microscopic study of the patterns of external root
resorption under different conditions